Final Technical Report on "Metabolic Engineering for Heavy Metal and Actinide Removal"

Complementary Z-Contrast Imaging and Digital Image Processing

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100118394 ◽

1985 ◽

Vol 43 ◽

pp. 304-305

Author(s):

K. N. Colonna ◽

G. Oliphant

Keyword(s):

Image Processing ◽

Heavy Metal ◽

Digital Image Processing ◽

Digital Image ◽

Biological Tissue ◽

Biological Imaging ◽

Tissue Preparation ◽

Contrast Imaging ◽

Imaging Tool ◽

Z Contrast

Harmonious use of Z-contrast imaging and digital image processing as an analytical imaging tool was developed and demonstrated in studying the elemental constitution of human and maturing rabbit spermatozoa. Due to its analog origin (Fig. 1), the Z-contrast image offers information unique to the science of biological imaging. Despite the information and distinct advantages it offers, the potential of Z-contrast imaging is extremely limited without the application of techniques of digital image processing. For the first time in biological imaging, this study demonstrates the tremendous potential involved in the complementary use of Z-contrast imaging and digital image processing.Imaging in the Z-contrast mode is powerful for three distinct reasons, the first of which involves tissue preparation. It affords biologists the opportunity to visualize biological tissue without the use of heavy metal fixatives and stains. For years biologists have used heavy metal components to compensate for the limited electron scattering properties of biological tissue.

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Recent Progress In High-Resolution Shadowing For Biological Transmission Electron Microscopy

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100181300 ◽

1990 ◽

Vol 48 (1) ◽

pp. 510-511 ◽

Cited By ~ 1

Author(s):

Heinz Gross ◽

Katarina Krusche ◽

Peter Tittmann

Keyword(s):

Heavy Metal ◽

High Resolution ◽

Freeze Drying ◽

Atmospheric Conditions ◽

Vacuum Equipment ◽

Transmission Electron ◽

Gas Atmosphere ◽

Gas Molecules ◽

Residual Gas ◽

Backing Layer

Freeze-drying followed by heavy metal shadowing is a long established and straight forward approach to routinely study the structure of dehydrated macromolecules. Very thin specimens such as isolated membranes or single macromolecules are directly adsorbed on C-coated grids. After rapid freezing the grids are transferred into a suitable vacuum equipment for freeze-drying and heavy metal shadowing.To improve the resolution power of shadowing films we introduced shadowing at very low specimen temperature (−250°C). To routinely do that without the danger of contamination we developed in collaboration with Balzers an UHV (p≤10-9 mbar) machine (BAF500K, Fig.2). It should be mentioned here that at −250°C the specimen surface acts as effective cryopump for practically all impinging residual gas molecules from the residual gas atmosphere.Common high resolution shadowing films (Pt/C, Ta/W) have to be protected from alterations due to air contact by a relatively thick C-backing layer, when transferred via atmospheric conditions into the TEM. Such an additional C-coat contributes disturbingly to the contrast at high resolution.

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Metabolic engineering of CHO cells to prepare glycoproteins

Emerging Topics in Life Sciences ◽

10.1042/etls20180056 ◽

2018 ◽

Vol 2 (3) ◽

pp. 433-442 ◽

Cited By ~ 1

Author(s):

Qiong Wang ◽

Michael J. Betenbaugh

Keyword(s):

Metabolic Engineering ◽

Cho Cells ◽

Genetic Manipulation ◽

Chinese Hamster ◽

Post Translational Modification ◽

Effector Functions ◽

Recombinant Glycoproteins ◽

Production Platform ◽

Chemical Inhibitors ◽

Amino Acid Mutations

As a complex and common post-translational modification, N-linked glycosylation affects a recombinant glycoprotein's biological activity and efficacy. For example, the α1,6-fucosylation significantly affects antibody-dependent cellular cytotoxicity and α2,6-sialylation is critical for antibody anti-inflammatory activity. Terminal sialylation is important for a glycoprotein's circulatory half-life. Chinese hamster ovary (CHO) cells are currently the predominant recombinant protein production platform, and, in this review, the characteristics of CHO glycosylation are summarized. Moreover, recent and current metabolic engineering strategies for tailoring glycoprotein fucosylation and sialylation in CHO cells, intensely investigated in the past decades, are described. One approach for reducing α1,6-fucosylation is through inhibiting fucosyltransferase (FUT8) expression by knockdown and knockout methods. Another approach to modulate fucosylation is through inhibition of multiple genes in the fucosylation biosynthesis pathway or through chemical inhibitors. To modulate antibody sialylation of the fragment crystallizable region, expressions of sialyltransferase and galactotransferase individually or together with amino acid mutations can affect antibody glycoforms and further influence antibody effector functions. The inhibition of sialidase expression and chemical supplementations are also effective and complementary approaches to improve the sialylation levels on recombinant glycoproteins. The engineering of CHO cells or protein sequence to control glycoforms to produce more homogenous glycans is an emerging topic. For modulating the glycosylation metabolic pathways, the interplay of multiple glyco-gene knockouts and knockins and the combination of multiple approaches, including genetic manipulation, protein engineering and chemical supplementation, are detailed in order to achieve specific glycan profiles on recombinant glycoproteins for superior biological function and effectiveness.

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